Proteomic analysis of biological fluids

ABSTRACT

The invention concerns the identification of proteomes of biological fluids and their use in determining the state of maternal/fetal conditions, including maternal conditions of fetal origin, chromosomal aneuploidies, and fetal diseases associated with fetal growth and maturation. In particular, the invention concerns the identification of the proteome of amniotic fluid (multiple proteins representing the composition of amniotic fluid) and the correlation of characteristic changes in the normal proteome with various pathologic maternal/fetal conditions, such as intra-amniotic infection, or chromosomal defects.

This application is a continuation of U.S. application Ser. No.10/400,005, filed Mar. 25, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns the identification of proteomes of biologicalfluids and their use in determining the state of maternal/fetalconditions, including maternal conditions of fetal origin, chromosomalaneuploidies, and fetal diseases associated with fetal growth andmaturation. In particular, the invention concerns the identification ofthe proteome of amniotic fluid (multiple proteins representing thecomposition of amniotic fluid) and the correlation of characteristicchanges in the normal proteome with various pathologic maternal/fetalconditions, such as intra-amniotic infection, or chromosomal defects.

2. Description of the Related Art

Proteomics

The large-scale analysis of protein expression patterns is emerging asan important and necessary complement to current DNA cloning and geneprofiling approaches (Pandey and Mann, Nature 405:837-46 (2000)). DNAsequence information is helpful in deducing some structural andpotential protein modifications based on homology methods, but it doesnot provide information on regulation of protein function throughpost-translational modifications, proteolysis or compartmentalization.

Traditional gel-based methods, such as one- and two-dimensional gelelectrophoresis are useful for small-scale protein detection (<1,000proteins), but these require large sample quantity (Lilley K S, RazzaqA, Dupree P: Two-dimensional gel electrophoresis: recent advances insample preparation, detection and quantitation. Curr Opin Chem Biol.6(1):46-50, 2002). Approaches to overcome this limitation includematrix-assisted or surface-enhanced laser desorption/ionization (MALDIor SELDI) time-of-flight mass spectrometers that accurately generateprofiles showing the masses of proteins in a sample. These patterns orprofiles can be used to identify and monitor various diseases. Thesecond level of identification comes from coupling peptide mapping totandem mass spectrometry to generate amino acid sequence informationfrom peptide fragments. This can, for example, be achieved by couplingthe MALDI/SELDI or ESI to quadrupole time-of-flight MS (Qq-TOF MS). Thelatter method can also be used for quantification of specific peptides(ICAT technology).

Diagnosis of Pathologic Maternal/Fetal Conditions

There are numerous pathologic maternal and fetal conditions, such asintra-amniotic infection (IAI), preeclampsia, preterm delivery andlabor, and chromosomal aneuploidies, that may develop during pregnancyand compromise the well-being or, in some instances, threaten the lifeof the mother and/or the fetus or newborn. Early diagnosis of suchconditions is critical to allow timely treatment and intervention.Unfortunately, early diagnosis for most of these conditions is difficultbecause the clinical signs and symptoms occur late, and are oftennon-specific and inconsistent. For example, the clinical symptoms of IAItypically include maternal fever and leukocytosis, but these symptomsoften occur later and are neither sensitive nor specific. Thus, Gravettet al., Am. J. Obstet. Gynecol. 171:1660-7 (1994), utilizing a non-humanprimate model, demonstrated that following experimental intra-amnioticinfection with Group B streptococcus, fever and leukocytosis are presentonly 50% of the time at the onset of infection-induced preterm labor,which occurs 28 to 40 hours after experimental infection. Therefore, toavoid a delay in diagnosis, a high index of suspicion and theappropriate use of adjunctive laboratory tests, are warranted. Theclinical criteria commonly used to diagnose IAI include maternal fever(≧37.8° C.), along with two or more of the following: maternalleukocytosis (≧15,000/mm³), maternal or fetal tachycardia, uterinetenderness, or foul-smelling amniotic fluid.

Because of the inconsistency of clinical features, other adjunctivelaboratory tests have been utilized to aid in the diagnosis of IAI.These include: measurement of maternal C-reactive protein, directexamination of amniotic fluid for leukocytes or bacteria on Gram stain,amniotic fluid culture, measurement of amniotic fluid glucoseconcentrations, detection of amniotic fluid leukocyte esterase,detection of bacterial organic acids by gas-liquid chromatography,measurements of various amniotic fluid or vaginal cytokines (e.g.,interleukins 2, 4, 6, granulocyte colony-stimulating factor, and tumornecrosis factor-α), matrix metalloproteinase-9, lactoferrin, andassessment of fetal activity (biophysical profile) by ultrasonography.Measurement of cytokines or other biochemical factors is expensive,generally not clinically available, and is primarily a research tool.Further, the testing efficiency of these tests has not been consistentlybetter than more readily available traditional tests such as amnioticfluid Gram stain and culture, amniotic fluid glucose concentrations, anddetection of amniotic fluid leukocyte esterase. The efficiency of thesetests has been previously extensively reviewed. (Ohlsson, A. and Wang,E.: An analysis of antenatal tests to detect infection at pretermrupture of the membranes. American Journal of Obstetrics and Gynecology162:809, 1990). Although all have reasonable sensitivity, specificity,and predictive value none are sufficiently sensitive or specific to beutilized independently of clinical features in the diagnosis of IAI.

Accordingly, there is a great need for new approaches that allow earlyand accurate diagnosis of IAI and other pathologic maternal/fetalconditions.

It is particularly desirable to develop new, efficient and reliablenon-invasive methods for the diagnosis of chromosomal aneuploidies. Atpresent the definitive diagnosis of chromosomal aneuploidies followingmaternal serum screening and ultrasound requires a mid-trimester geneticamniocentesis. This is an invasive procedure associated with a 0.5% riskof loss of the pregnancy. Further, chromosomal analysis of amnioticfluid cells is a labor-intensive and time-consuming procedure, taking upto 2 weeks. Reliable tests are therefore necessary to improve thedetection of chromosomal aneuploidies from maternal serum, or otherbiological fluids, reduce the unacceptably high false positive rate ofmaternal screening, and increase the speed and efficiency of diagnosisfrom amniotic fluid following amniocentesis. Other pathologicaneuploidic conditions, such as Klinefelter syndrome and Turnersyndrome, may be entirely missed by screening with ultrasonography orconventional maternal serum screening.

SUMMARY OF THE INVENTION

The present invention provides non-invasive and sensitive methods forthe early diagnosis, prognosis, and monitoring of pathologicfetal/maternal conditions, by proteomic analysis of biological fluids.

The present invention further provides proteomic profiles of biologicalfluids, such as amniotic fluid and maternal serum, which enable thediagnosis, prognosis, and monitoring of various pathologicfetal/maternal conditions, including, without limitation, intra-amnioticinfection (IAI), chromosomal aneuploidies, and fetal diseases associatedwith fetal growth and maturation. In particular, the present inventionprovides normal and pathologic proteomic profiles for IAI andchromosomal aneuploidies. The determination of the normal proteomicprofile is of great importance, since it enables the elimination of thefetal/maternal condition in question (negative diagnosis), whicheliminates the need to subject the patient to unnecessary andpotentially dangerous treatment or intervention.

The present invention further provides specific biomarkers for thepresence and state of IAI and chromosomal aneuploidies, which aredifferentially expressed in biological fluids, such as amniotic fluid ormaternal serum, when such pathologic conditions are present.

In one aspect, the invention concerns a method for determining the stateof a maternal or fetal condition, comprising comparing the proteomicprofile of a test sample of a biological fluid obtained from a mammaliansubject with the proteomic profile of a normal sample, or a referenceproteomic profile comprising at least one unique expression signaturecharacteristic of such condition.

In an embodiment of this method, the mammalian subject is a pregnantfemale, preferably primate or human.

In another embodiment, the maternal condition is selected from the groupconsisting of intrauterine infection, preeclampsia, and preterm labor.

In a further embodiment, the fetal condition is selected from the groupconsisting of chromosomal aneuploidies, congenital malformation,gestational age and fetal maturity, where the chromosomal aneuploidycan, for example, be Down syndrome, trisomy-13, trisomy-18, Turnersyndrome, or Klinefelter syndrome.

Any biological fluid can be used in performing the method of theinvention, including, without limitation, amniotic fluid, serum, plasma,urine, cerebrospinal fluid, breast milk, mucus, and saliva, preferably,amniotic fluid or maternal serum.

In a further embodiment, the proteomic profile of the test samplecomprises information of at least 2 proteins, or at least 5 proteins, orat least 10 proteins, or at least 20 proteins, or at least 50 proteins.

In a specific embodiment, the proteomic profile is a mass spectrum.

In another embodiment, the mass spectrum comprises at least one uniqueexpression signature in the 3 to 5 kDa range of the mass spectrum.

In yet another embodiment, the mass spectrum comprises at least oneunique expression signature in the 10 to 12 kDa range of the massspectrum.

In a further embodiment, the maternal condition is intra-amnioticinfection, and the unique expression signature is an extra peak in the10 to 11 kDa molecular weight range in the test sample, which isindicative of intra-amniotic infection.

In a different embodiment, the proteomic profile is produced by Westernblot analysis.

In another embodiment, the biological fluid is that of a human, and theproteomic profile includes information of the expression of one or moreof the proteins selected from the group consisting of: macrophagecapping protein, neutrophil gelatinase-associated lipocalin,myeloperoxidase; L-plastin; azurocidin; antibacterial protein FALL-39;Gp340 variant protein; Ebner salivary gland protein homologoue (GenBankAccession No. 355392); leukocyte elastase inhibitor; calgranulin A;calgranulin B; cofilin; moesin; profilin I, cronin-like protein p57;annexin II, fibronectin; glia-derived nexin; antithrombin-III; squamouscell carcinoma antigen 1, squamous cell carcinoma antigen 2; serpin 12;cystatin A; cystatin B; cystatin C; IGFBP-1; Vitamin D-binding protein;apolipoprotein A-I; 14-3-3 protein sigma; 14-3-3 protein zeta/delta;gelsolin; lactotransferrin; phosphoglycerate kinase 1; phosphoglyceratemutase 1; and transketolase; or a fragment, precursor, or naturallyoccurring variant thereof.

In a further embodiment, the proteomic profile includes information ofthe expression of one or more of the proteins selected from the groupconsisting of macrophage capping protein; neutrophilgelatinase-associated lipocalin; myeloperoxidase; L-plastin; azurocidin;antibacterial protein FALL-39; leukocyte elastase inhibitor; calgranulinA; calgranulin B; profilin I, glia-derived nexin; serpin 12; cystatin A;and IGFBP-1; or a fragment, precursor, or naturally occurring variantthereof.

The foregoing method is suitable for the diagnosis of various fetal andmaternal conditions, including, without limitation, intra-amnioticinfection, developmental defects, including defects of an organ system,musculoskeletal deformities, and conditions resulting from chromosomalaneuploidies, such as Down syndrome, trisomy-13, trisomy-18, Turnersyndrome, or Klinefelter syndrome.

If the proteomic profile of the test sample is essentially the same asthe proteomic profile of the normal sample, the subject is determined tobe free of the maternal or fetal condition.

If the proteomic profile contains essentially the same unique expressionsignature as a diseased sample, the patient is diagnosed with thecorresponding maternal or fetal condition.

In another aspect, the invention concerns a method for the diagnosis ofintra-amniotic infection, comprising

(a) comparing the proteomic profile of a test sample of a biologicalfluid obtained from a pregnant female mammal with the proteomic profileof a normal sample, or a reference proteomic profile, wherein theproteomic profiles provide information of the mass of the proteinspresent in the samples, or the proteolytic fragments thereof; and

(b) diagnosing the mammal with intra-amniotic infection if the proteomicprofile of the test sample shows a unique expression signature in the3-5 and/or 10-12 KDa molecular weight range.

In a further aspect, the invention concerns a method for the diagnosisof intra-amniotic infection, comprising:

(a) comparing the proteomic profile of a test sample of a biologicalfluid obtained from a pregnant female mammal with the proteomic profileof a normal sample; and

(b) diagnosing the mammal with intra-amniotic infection if at least oneprotein selected from the group consisting of IGFB-1, profilin,ceruloplasmin, L-plastin, and calgraulin, or a fragment, precursor ornaturally occurring variant thereof, is differentially expressed in thetest sample relative to the normal sample.

In a particular embodiment, at least one of IGFBP-1, profilin,ceruloplasmin, and calgranulin, or a fragment, precursor, ornaturally-occurring variant thereof, is overexpressed in the test samplerelative to the normal sample.

In another embodiment, L-plastin is underexpressed in the test samplerelative to the normal sample.

In yet another embodiment, the presence of IGFBP-1 is detected byidentifying the proteolytic fragment shown in FIG. 12, or a fragmentthereof.

In another aspect, the invention concerns a method for the diagnosis ofa chromosomal aneuploidy, comprising:

(a) comparing the proteomic profile of a test sample of a biologicalfluid obtained from a pregnant female mammal with the proteomic profileof a normal sample, or a reference proteomic profile, wherein theproteomic profiles provide information of the mass of the proteinspresent in the samples, or the proteolytic fragments thereof; and

(b) diagnosing the mammal with the chromosomal aneuploidy if theproteomic profile of the test sample shows a unique expression signaturein the 4 to 15 KDa molecular weight range.

In a different aspect, the invention concerns a method for the diagnosisof a developmental defect of a fetus, comprising:

(a) comparing the proteomic profile of a test sample of a biologicalfluid obtained from a pregnant female mammal with the proteomic profileof a normal sample, or a reference proteomic profile; and

(b) confirming the presence of the developmental defect if at least oneactin-modulating protein, or a fragment, precursor, or naturallyoccurring variant thereof, is differentially expressed in the testsample relative to the normal sample.

In a particular embodiment of this method, the actin-modulating proteinis selected from the group consisting of moesin, p57, gelsolin, and a14-3-3 protein.

In a further aspect, the invention concerns a method for the diagnosisof a maternal or fetal infection or immune-response related disorder,comprising

(a) comparing the proteomic profile of a test sample of a biologicalfluid obtained from a pregnant female mammal with the proteomic profileof a normal sample, or a reference proteomic profile; and

(b) confirming the presence of the maternal or fetal infection orimmune-response related disorder, if at least one protein selected fromthe group consisting of macrophage capping protein (MCP), leukocyteelastase, neutrophil gelatinase-associated lipcalcin (NGAL),myeloperoxidase, L-plastin, calgranulin, FALL-39, azyrocidin (CAP37),proteases and protease inhibitors, is differentially expressed in thetest sample relative to the normal sample.

In a still further aspect, the invention concerns a method for thediagnosis of neonatal sepsis, comprising detecting in the proteomicprofile of a biological fluid obtained from a pregnant females mammalthe presence of Gp-340.

In yet another aspect, the invention concerns a proteomic profile of abiological fluid comprising information of one or more proteins selectedfrom the group consisting of macrophage capping protein, neutrophilgelatinase-associated lipocalin, myeloperoxidase; L-plastin; azurocidin;antibacterial protein FALL-39; Gp340 variant protein; Ebner salivarygland protein homologoue (GenBank Accession No. 355392); leukocyteelastase inhibitor; calgranulin A; calgranulin B; cofilin; moesin;profilin I, cronin-like protein p57; annexin II, fibronectin;glia-derived nexin; antithrombin-III; squamous cell carcinoma antigen 1,squamous cell carcinoma antigen 2; serpin 12; cystatin A; cystatin B;cystatin C; IGFBP-1; Vitamin D-binding protein; apolipoprotein A-I;14-3-3 protein sigma; protein zeta/delta; gelsolin; lactotransferrin;phosphoglycerate kinase 1; phosphoglycerate mutase 1; and transketolase;or a fragment, precursor, or naturally occurring variant thereof.

In a further aspect, the invention concerns a proteomic profile of abiological fluid comprising information of one or more proteins selectedfrom the group consisting of macrophage capping protein; neutrophilgelatinase-associated lipocalin; myeloperoxidase; L-plastin; azurocidin;antibacterial protein FALL-39; leukocyte elastase inhibitor; calgranulinA; calgranulin B; profilin I, glia-derived nexin; serpin 12; cystatin A;and IGFBP-1; or a fragment, precursor, or naturally occurring variantthereof.

The invention further concerns a proteomic profile of a biological fluidcharacteristic of intra-amniotic infection, comprising informationconfirming the presence of a protein selected from the group consistingof IGFB-1, profilin, ceruloplasmin, L-plastin, and calgraulin.

In another aspect, the invention concerns a proteomic profile of abiological fluid characteristic of intra-amniotic infection representedin a form providing information of the molecular weight of proteinspresent in the biological fluid, or the proteolytic fragments thereof,comprising a unique expression signature in the 3-5 KDa and/or 10-12 KDamolecular weight range.

In a further aspect, the invention concerns the proteomic profileessentially as shown in any one of FIGS. 1A-1C, or essentially as shownin any one of FIGS. 2A-C, or essentially as shown in any one of FIGS.3A-C, or essentially as shown in FIG. 4A or 4B, or essentially as shownin any one of FIGS. 6-10.

In a particular embodiment, the proteomic profile is analyzed in amicroarray format.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C. Infection-induced differential protein expression in theprimate amniotic fluid. SELDI-TOF analysis of amniotic fluid extractsbound to chemically defined Normal Phase chip arrays. A). Whole spectrumcollected at 235 laser intensity showing the differences in the peakintensities. B) Detailed spectrum showing the differences in the 10 to12 KDa region between control and infected. C) Detailed spectrum showingthe differences in the 3-5 KDa region between control and infected.Solid lines were used to show the significant differences in expression(unique expression signatures) which could be used to develop diagnostictests.

FIGS. 2A-C. Time course analyses of the primate amniotic fluid inresponse to infection (GBS). Amniotic fluid was collected before theinoculation of bacteria and serially after infection and subjected toSELDI-TOF analysis as described below. FIG. 2 A: before infection; 2B:12 hours after infection; 2C: 36 hours after infection.

FIGS. 3A-C. Infection-induced differential protein expression in thehuman amniotic fluid. SELDI-TOF analysis of amniotic fluid extractsbound to chemically defined Normal Phase chip arrays. A). Whole spectrumcollected at 235 laser intensity showing the differences in the peakintensities. B) Detailed spectrum showing the differences in the 10 to12 KDa region between control and infected. C) Detailed spectrum showingthe differences in the 3-5 KDa region between control and infected.

FIGS. 4A and 4B. Mass spectra acquired on a generic MALDI-TOF massspectrometer, using amniotic fluid from human A) control, withoutintrauterine infection and B) sample, with intrauterine infection.

FIG. 5. SDS-PAGE Commassie Blue stained gel. A) 4 human control AFsamples pooled; B) individual control AF sample; C) 4 human infected AFsamples pooled D) individual infected AF sample.

FIG. 6. Detection of differential protein expression in the humanamniotic fluid. A) Control AF sample (pooled); B) Infected AF sample(pooled).

FIG. 7. Detection of differential protein expression in the humanamniotic fluid. A) Control AF sample (pooled); B) Infected AF sample(pooled).

FIG. 8 shows the detection of differential protein expression in thehuman amniotic fluid and maternal serum. A) control sample (pooled); B)infected sample (pooled).

FIG. 9 shows the detection of differentially expressed proteins inmaternal serum using protein arrays. 1) pseudocolor image of the proteinarray showing the binding of corresponding proteins with theirantibodies; 2) enlarged area of the array; 3) Western blot ofcalgranulin IP.

FIG. 10 shows differential protein expression patterns in the maternalserum with unique profiles to distinguish trisomies.

FIG. 11. Schematic representation of de novo protein sequenceidentification of amniotic fluid proteins. PRO1_HUMAN (P07737) ProfilinI (SEQ ID Nos: 5-11).

FIG. 12. IGFBP-1 de novo protein identification and proteolytic fragmentsequence (SEQ ID No: 1). The peptide sequences found in samples0426se_H1_(—)12 and 0425se_H1_(—)13 with the Ms/MS are shown in lowercase (SEQ ID Nos: 2 and 3). These came from infected amniotic fluid whenrun on 1-D gel bands that were trypsin digested and subjected to MS/MSanalysis. The proteolytic fragment of IGF-BP-1 detected in 1-D gels (lowmolecular weight range, FIG. 5), Western blots (FIG. 6) and MS/MSanalysis (FIG. 13) of trypsin-digested ˜10.5 to 12 KDa band frominfected amniotic fluid is represented in the region of the underlinedsequence (SEQ ID No: 4).

FIG. 13. LCQ-MS profile of trypsin digestion of 10.5-11 kD ID gel bandfrom infected amniotic fluid. LCQ-MS showing parent ions representingpotential proteins present in the sample.

FIG. 14. Mass spectrum for the 17.55-18.21 minute retention time peakshown in FIG. 13.

FIG. 15. MS/MS spectrum for the parent ion of the 434.9 peak shown inFIG. 14.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994) provides one skilled in the art with a general guide to manyof the terms used in the present application.

The term “proteome” is used herein to describe a significant portion ofproteins in a biological sample at a given time. The concept of proteomeis fundamentally different from the genome. While the genome isvirtually static, the proteome continually changes in response tointernal and external events.

The term “proteomic profile” is used to refer to a representation of theexpression pattern of a plurality of proteins in a biological sample,e.g. a biological fluid at a given time. The proteomic profile can, forexample, be represented as a mass spectrum, but other representationsbased on any physicochemical or biochemical properties of the proteinsare also included. Thus the proteomic profile may, for example, be basedon differences in the electrophoretic properties of proteins, asdetermined by two-dimensional gel electrophoresis, e.g. by 2-D PAGE, andcan be represented, e.g. as a plurality of spots in a two-dimensionalelectrophoresis gel. Differential expression profiles may have importantdiagnostic value, even in the absence of specifically identifiedproteins. Single protein spots can then be detected, for example, byimmunoblotting, multiple spots or proteins using protein microarrays.The proteomic profile typically represents or contains information thatcould range from a few peaks to a complex profile representing 50 ormore peaks. Thus, for example, the proteomic profile may contain orrepresent at least 2, or at least 5 or at least 10 or at least 15, or atleast 20, or at least 25, or at least 30, or at least 35, or at least40, or at least 45, or at least 50 proteins.

The term “pathologic condition” is used in the broadest sense and coversall changes and phenomena that compromise the well-being of a subject.Pathologic maternal conditions include, without limitation,intra-amniotic infection, conditions of fetal or maternal origin, suchas, for example preeclampsia, and preterm labor and delivery. Pathologicfetal conditions include, without limitation, chromosomal defects(aneuploidies), such as Down syndrome, and all abnormalities ingestational age and fetal maturity.

The term “state of a pathologic [maternal or fetal] condition” is usedherein in the broadest sense and refers to the absence, presence,extent, stage, nature, progression or regression of the pathologiccondition.

The term “unique expression signature” is used to describe a uniquefeature or motif within the proteomic profile of a biological sample(e.g. a reference sample) that differs from the proteomic profile of acorresponding normal biological sample (obtained from the same type ofsource, e.g. biological fluid) in a statistically significant manner.

The terms “intra-amniotic infection (IAI),” “amniotic fluid infection,”“amnionitis,” and “clinical chorioamnionitis” are used interchangeably,and refer to an acute infection, including, but not restricted tobacterial, of the amniotic fluid and intrauterine contents duringpregnancy.

“Patient response” can be assessed using any endpoint indicating abenefit to the patient, including, without limitation, (1) inhibition,at least to some extent, of the progression of a pathologic condition,(2) prevention of the pathologic condition, (3) relief, at least to someextent, of one or more symptoms associated with the pathologiccondition; (4) increase in the length of survival following treatment;and/or (5) decreased mortality at a given point of time followingtreatment.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathologic condition or disorder.Those in need of treatment include those already with the disorder aswell as those prone to have the disorder or those in whom the disorderis to be prevented.

“Congenital malformation” is an abnormality which is non-hereditary butwhich exists at birth.

B. Detailed Description

The present invention concerns methods and means for an early, reliableand non-invasive testing of maternal and fetal conditions based upon theproteomic profile of a biological fluid of the mother or fetus. Theinvention utilizes proteomics techniques well known in the art, asdescribed, for example, in the following textbooks, the contents ofwhich are hereby expressly incorporated by reference: Proteome Research:New Frontiers in Functional Genomics (Principles and Practice), M. R.Wilkins et al., eds., Springer Verlag, 1007; 2-D Proteome AnalysisProtocols, Andrew L Link, editor, Humana Press, 1999; Proteome ResearchTwo-Dimensional Gel Electrophoresis and Identification Methods(Principles and Practice), T. Rabilloud editor, Springer Verlag, 2000;Proteome Research: Mass Spectrometry (Principles and Practice), P. Jameseditor, Springer Verlag, 2001; Introduction to Proteomics, D. C. Lieblereditor, Humana Press, 2002; Proteomics in Practice: A Laboratory Manualof Proteome Analysis, R. Westermeier et al., eds., John Wiley & Sons,2002.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

1. Identification of Proteins and Polypeptides Expressed in BiologicalFluids

According to the present invention, proteomics analysis of biologicalfluids can be performed using a variety of methods known in the art.

Typically, protein patterns (proteome maps) of samples from differentsources, such as normal biological fluid (normal sample) and a testbiological fluid (test sample), are compared to detect proteins that areup- or down-regulated in a disease. These proteins can then be excisedfor identification and full characterization, e.g. using peptide-massfingerprinting and/or mass spectrometry and sequencing methods, or thenormal and/or disease-specific proteome map can be used directly for thediagnosis of the disease of interest, or to confirm the presence orabsence of the disease.

In comparative analysis, it is important to treat the normal and testsamples exactly the same way, in order to correctly represent therelative abundance of proteins, and obtain accurate results. Therequired amount of total proteins will depend on the analyticaltechnique used, and can be readily determined by one skilled in the art.The proteins present in the biological samples are typically separatedby two-dimensional gel electrophoresis (2-DE) according to their pI andmolecular weight. The proteins are first separated by their charge usingisoelectric focusing (one-dimensional gel electrophoresis). This stepcan, for example, be carried out using immobilized pH-gradient (IPG)strips, which are commercially available. The second dimension is anormal SDS-PAGE analysis, where the focused IPG strip is used as thesample. After 2-DE separation, proteins can be visualized withconventional dyes, like Coomassie Blue or silver staining, and imagedusing known techniques and equipment, such as, e.g. Bio-Rad GS800densitometer and PDQUEST software, both of which are commerciallyavailable. Individual spots are then cut from the gel, destained, andsubjected to tryptic digestion. The peptide mixtures can be analyzed bymass spectrometry (MS). Alternatively, the peptides can be separated,for example by capillary high pressure liquid chromatography (HPLC) andcan be analyzed by MS either individually, or in pools.

Mass spectrometers consist of an ion source, mass analyzer, iondetector, and data acquisition unit. First, the peptides are ionized inthe ion source. Then the ionized peptides are separated according totheir mass-to-charge ratio in the mass analyzer and the separate ionsare detected. Mass spectrometry has been widely used in proteinanalysis, especially since the invention of matrix-assistedlaser-desorption ionisation/time-of-flight (MALDI-TOF) and electrosprayionisation (ESI) methods. There are several versions of mass analyzer,including, for example, MALDI-TOF and triple or quadrupole-TOF, or iontrap mass analyzer coupled to ESI. Thus, for example, a Q-Tof-2 massspectrometer utilizes an orthogonal time-of-flight analyzer that allowsthe simultaneous detection of ions across the full mass spectrum range.For further details see, e.g. Chemusevich et al., J. Mass Spectrom.36:849-865 (2001).

If desired, the amino acid sequences of the peptide fragments andeventually the proteins from which they derived can be determined bytechniques known in the art, such as certain variations of massspectrometry, or Edman degradation.

2. Fetal-Maternal Conditions Benefiting from Early and Non-InvasiveDiagnosis

a. Intra-Amniotic Infection

Intra-amniotic infection (IAI) is an acute bacterial infection of theamniotic fluid and intrauterine contents during pregnancy. Prospectivestudies indicate that IAI occurs in 4% to 10% of all deliveries (Newton,E. R., Prihoda, T. J., and Gibbs, R. S.: Logistic regression analysis ofrisk factors for intra-amniotic infection. Obstet. Gynecol. 73:571,1989; Soper, D. E., Mayhall, C. G., and Dalton, H. P.: Risk factors forintraamniotic infection: a prospective epidemicologic study. AmericanJournal of Obstetrics and Gynecology 161:562, 1989; and Lopez-Zeno, J.A., Peaceman, A. M., Adashek, J. A., and Socol, M. L.: A controlledtrial of a program for the active management of labor. N. Engl. J. Med.326:450, 1992). Other terms used to describe IAI include amniotic fluidinfection, amnionitis, and clinical chorioamnionitis. Intra-amnioticinfection is clinically diagnosed by maternal fever, uterine tenderness,leukocytosis, and fetal tachycardia and should be distinguished fromhistologic chorioamnionitis. Intra-amniotic infection is an importantcause of maternal and neonatal morbidity. Intra-amniotic infectionaccounts for 10-40% of cases of febrile morbidity in the peripartumperiod and is associated with 20-40% of cases of early neonatal sepsisand pneumonia (Newton, E. R.: Chorioamnionitis and intraamnioticinfection. Clin. Obstet. Gynecol. 36:795, 1993). Maternal bacteremiaoccurs in 2-6% of patients with IAI and postpartum infectious morbidityis increased. There is also an increased risk of dysfunctional labor andcesarean delivery among patients with IAI. Duff et al. reported a 75%incidence of dysfunctional labor and a 34% incidence of cesareandelivery among patients who developed intra-amniotic infection while inlabor (Duff, P., Sanders, R., and Gibbs, R. S.: The course of labor interm pregnancies with chorioamnionitis. American Journal of Obstetricsand Gynecology 147:391, 1983). Intra-amniotic infection is alsoassociated with increased neonatal morbidity and mortality, particularlyamong preterm neonates. In general, there is a three to four-foldincrease in perinatal mortality among low birth weight neonates born tomothers with IAI (Gibbs, R. S., Castillo, M. A., and Rodgers, P. J.:Management of acute chorioamnionitis. American Journal of Obstetrics andGynecology 136:709, 1980; Gilstrap, L. C., III, Leveno, K. J., Cox, S.M., Burris, J. S., Mashburn, M., and Rosenfeld, C. R.: Intrapartumtreatment of acute chorioamnionitis: impact on neonatal sepsis. Am. J.Obstet. Gynecol. 159:579, 1988). There are also increases in respiratorydistress syndrome, intraventricular hemorrhage, and neonatal sepsisMorales, W. J.: The effect of chorioamnionitis on the developmentaloutcome of preterm infants at one year. Obstetrics and Gynecology70:183, 1987). Recently, IAI has been implicated in neonatalperiventricular leukomalacia and cerebral palsy; the risks of cerebralwhite matter damage and cerebral palsy are nine-fold greater in thesetting of intra-amniotic infection Bejar, R., Wozniak, P., Allard, M.,Benirschke, K., Vaucher, Y., Coen, R., Berry, C., Schragg, P., Villegas,I., and Resnik, R.: Antenatal origin of neurologic damage in newborninfants. I. Preterm infants. Am. J. Obstet. Gynecol. 159:357, 1988;Grether, J. K. and Nelson, K. B.: Maternal infection and cerebral palsyin infants of normal birth weight. JAMA 278:207, 1997). Finally,subclinical IAI has been found in at least 10% of women in preterm laborwith intact fetal membranes, suggesting that IAI is an important, andpotentially preventable, cause of prematurity (Romero, R., Avila, C.,Brekus, C. A., and Morotti, R.: The role of systemic and intrauterineinfection in preterm parturition. Annuals of the New York Academy ofSciences 622:355, 1991). A literature review by Newton demonstratedincidences of clinical IAI of 41% at gestational ages less than 27weeks, 15% at gestational ages of 27-37 weeks, and 2% at gestations of38 weeks or greater (Newton et al., supra). Bacteria indigenous to thelower genital tract have also been recovered from the amniotic fluid of10-20% of all women in preterm labor with intact fetal membranes withoutclinical signs of intraamniotic infection (Romero et al., supra), and inup to 67% of women in preterm labor with pregnancies ending at 23-24weeks (Watts, D. H., Krohn, M. A., Hillier, S. L., and Eschenbach, D.A.: The association of occult amniotic fluid infection with gestationalage and neonatal outcome among women in preterm labor. Obstet Gynecol79:351, 1992). Most of these patients deliver rapidly, and clinicallyapparent IAI develops in many. These observations support the hypothesisthat ascending, initially subclinical intrauterine infections-precedepreterm labor and may be an important cause of extreme pretermdeliveries.

b. Preeclampsia

Preeclampsia, defined as maternal hypertension accompanied byproteinuria, edema, or both, occurs in 7% of pregnancies not terminatingin the first trimester. Although the cause is unknown, it is more commonin extremes of age in childbearing, maternal diabetes, pregnancies withmultiple gestations, and pre-existing maternal renal disease and orhypertension. Preeclampsia is associated with increases in perinatalmortality, and may also lead to eclampsia, characterized by maternalseizures and increased maternal mortality. Currently the mainstay oftherapy for preeclampsia is delivery and anticonvulsant prophylaxis withmagnesium sulfate. Prior to the advent of magnesium sulfate therapy, theobserved maternal mortality was 20-30%. However, with prompt diagnosis,allowing anticonvulsant therapy with magnesium sulfate,anti-hypertensives, and delivery the maternal mortality has been reducedto near zero.

Unfortunately, the diagnosis of preeclampsia based upon commonlyrecognized symptoms and signs is frequently difficult, and occurs latein the course of the disease. Frequently fetal compromise in growth orwell-being is the first recognized manifestation of preeclampsia.Laboratory markers for preeclampsia include quantitation of proteinuria,and elevated serum concentrations of uric acid or creatinine. There areno currently available serum markers for early preeclampsia or markerswhich identify women which will develop preeclampsia. Recentlyprospective serum markers including leptin and uric acid have beenassociated with subsequent preeclampsia in one study (Gursoy T, et al.Preeclampsia disrupts the normal physiology of leptin: Am J Perinatol.19(6):303-10, 2002) but much work is needed to confirm these findings.Development of early and reliable markers for preeclampsia is imperativeto allow for therapy and intervention to optimize the outcome for theneonate and mother.

c. Preterm Labor and Delivery

Preterm delivery is defined as birth prior to the 37^(th) completed weekof gestation. The incidence of preterm birth in the United States is10-11% of all live births, and is increasing despite aggressivetreatment of preterm labor. Overall, prematurity and its consequencesare responsible for 80% of perinatal deaths not attributable tocongenital malformations and add approximately $5 billion annually tothe national health care budget. Risk factors for preterm birth includenon-white race, young age, low socioeconomic status, maternal weightbelow 55 kg, nulliparity, 1^(st) trimester bleeding, multiple gestations(Meis P J, Michielutte R, Peters T J, et al. Factors associated withpreterm birth in Cardiff, Wales: II. Indicated and spontaneous pretermbirth. Am J Obstet Gynecol 173:597-602, 1995)

Unfortunately the prediction of patients at risk for spontaneous pretermbirth has been generally disappointing (Creasy R K, lams JD. Pretermlabor and delivery. In Maternal-Fetal Medicine, Creasy R K, Resnik R(eds.). W.B. Saunders Company, Philadelphia, Pa. 4^(th) edition, 1999.Pages 498-531). Previous attempts at defining the population at greatestrisk for preterm birth, and thereby potentially benefiting from earlyintervention have included risk-scoring indices, biochemical detectionof cervical fetal fibronectin, ultrasound measurement of cervicallength, and home uterine activity monitoring. These programs have beenboth costly, and have been hampered by the inability to predict withaccuracy which patients might benefit from early intervention orprophylaxis. All suffer from poor positive predictive value ofapproximately 30%, with the majority of patients identified as “at risk”delivering at term. Interventions, including pharmacologic treatment toinhibit uterine contractions, are efficacious, but depend upon the earlyand reliable diagnosis of preterm labor. Early and reliable markers toidentify patients at greatest risk for preterm birth are thereforenecessary to reduce the tremendous costs and neonatal mortality andmorbidity associated with preterm birth.

d. Chromosomal Aneuploidies

Chromosomal abnormalities are a frequent cause of perinatal morbidityand mortality. Chromosomal abnormalities occur with an incidence of 1 in200 live births. The major cause of these abnormalities is chromosomalaneuploidy, an abnormal number of chromosomes inherited from theparents. One of the most frequent chromosomal aneuploidies is trisomy-21(Down syndrome), which has an occurrence of 1 in 800 livebirths (Hook EB, Hamerton J L: The frequency of chromosome abnormalities detected inconsecutive newborn studies: Differences between studies: Results by sexand by severity of phenotypic involvement. In Hook E B, Porter I H(eds): Population Cytogenetics, pp 63-79. New York, Academic Press,1978). The primary risk factor for trisomy-21 is maternal age greaterthan 35, but 80% of children with trisomy-21 are born to women youngerthan 35 years of age. Other common aneuploidic conditions includetrisomies 13 and 18, Turner Syndrome and Klinefelter syndrome.

Because 80% of children with trisomy-21 are born to women younger than35 years of age, prenatal diagnostic screening programs designed on thebasis of maternal age alone are inefficient. Prenatal screening programshave therefore been supplemented with maternal serum screening foranalytes associated with fetal chromosomal aneuploidy, ultrasound, or acombination of both. Candidate serum markers that have been widelyutilized include alpha-fetoprotein (AFP), unconjugated estriol, humanchoriogonadotrophic hormone (hHCG), and inhibin-A. However, with ascreen positive rate of 2-5%, the detection rate for trisomy-21 andother aneuploidies has been disappointing, with detection rates of only70-86% (Cuckle H. Biochemical screening for Down syndrome. Eur J ObstetGynecol Reprod Biol. 92(1):97-101, 2000). Further, the rate of truepositive tests, i.e., trisomy-21 among those with a screen positive testis only 1-2%, resulting in an overall false positive rate in excess of98%.

The definitive diagnosis of chromosomal aneuploidies following maternalserum screening and ultrasound requires a mid-trimester geneticamniocentesis. This is an invasive procedure associated with a 0.5% riskof loss of the pregnancy. Further, chromosomal analysis of amnioticfluid cells is a labor-intensive and time consuming procedure, taking upto 2 weeks. Reliable tests are therefore necessary to improve thedetection of chromosomal aneuploidies from maternal serum, reduce theunacceptably high false positive rate of maternal screening, andincrease the speed and efficiency of diagnosis from amniotic fluidfollowing amniocentesis.

3. Diagnosis of Maternal/Fetal Conditions Using the Proteomic Profile ofBiological Fluids

The present invention provides an early and reliable, non-invasivemethod for the diagnosis of the foregoing and other similarmaternal/fetal conditions by proteomic analysis of biological fluids,such as, for example, amniotic fluid, serum, plasma, urine,cerebrospinal fluid, breast milk, mucus, or saliva.

As noted before, in the context of the present invention the term“proteomic profile” is used to refer to a representation of theexpression pattern of a plurality of proteins in a biological sample,e.g. a biological fluid at a given time. The proteomic profile can, forexample, be represented as a mass spectrum, but other representationsbased on any physicochemical or biochemical properties of the proteinsare also included. Although it is possible to identify and sequence allor some of the proteins present in the proteome of a biological fluid,this is not necessary for the diagnostic use of the proteomic profilesgenerated in accordance with the present invention. Diagnosis of aparticular disease can be based on characteristic differences (uniqueexpression signatures) between a normal proteomic profile, and proteomicprofile of the same biological fluid obtained under the samecircumstances, when the disease or pathologic condition to be diagnosedis present. The unique expression signature can be any unique feature ormotif within the proteomic profile of a test or reference biologicalsample that differs from the proteomic profile of a corresponding normalbiological sample obtained from the same type of source, in astatistically significant manner. For example, if the proteomic profileis presented in the form of a mass spectrum, the unique expressionsignature is typically a peak or a combination of peaks that differ,qualitatively or quantitatively, from the mass spectrum of acorresponding normal sample. Thus, the appearance of a new peak or acombination of new peaks in the mass spectrum, or any statisticallysignificant change in the amplitude or shape of an existing peak orcombination of existing peaks in the mass spectrum can be considered aunique expression signature. When the proteomic profile of the testsample obtained from a mammalian subject is compared with the proteomicprofile of a reference sample comprising a unique expression signaturecharacteristic of a pathologic maternal or fetal condition, themammalian subject is diagnosed with such pathologic condition if itshares the unique expression signature with the reference sample.

A particular pathologic maternal/fetal condition can be diagnosed bycomparing the proteomic profile of a biological fluid obtained from thesubject to be diagnosed with the proteomic profile of a normalbiological fluid of the same kind, obtained and treated the same manner.If the proteomic profile of the test sample is essentially the same asthe proteomic profile of the normal sample, the subject is considered tobe free of the subject pathologic maternal/fetal condition. If theproteomic profile of the test sample shows a unique expression signaturerelative to the proteomic profile of the normal sample, the subject isdiagnosed with the maternal/fetal condition in question.

Alternatively or in addition, the proteomic profile of the test samplemay be compared with the proteomic profile of a reference sample,obtained from a biological fluid of a subject independently diagnosedwith the pathologic maternal/fetal condition ion question. In this case,the subject is diagnosed with the pathologic condition if the proteomicprofile of the test sample shares at least one feature, or a combinationof features representing a unique expression signature, with theproteomic profile of the reference sample.

In the methods of the present invention the proteomic profile of anormal biological sample plays an important diagnostic role. Asdiscussed above, if the proteomic profile of the test sample isessentially the same as the proteomic profile of the normal biologicalsample, the patient is diagnosed as being free of the pathologicmaternal/fetal condition to be identified. This “negative” diagnosis isof great significance, since it eliminates the need of subjecting apatient to unnecessary treatment or intervention, which could havepotential side-effects, or may otherwise put the patient, fetus, orneonate at risk. The data are analyzed to determine if the differencesare statistically significant.

The sensitivity of the diagnostic methods of the present invention canbe enhanced by removing the proteins found both in normal and diseasedproteome at essentially the same expression levels (common proteins,such as albumin and immunoglobulins) prior to analysis usingconventional protein separation methods. The removal of such commonproteins, which are not part of the unique expression signature, resultsin improved sensitivity and diagnostic accuracy. Alternatively or inaddition, the expression signatures of the common proteins can beeliminated (or signals can be removed) during computerized analysis ofthe results, typically using spectral select algorithms, that aremachine oriented, to make diagnostic calls.

The results detailed in the Examples below present proteomic profilescharacteristics of intraamniotic infection (IAI) that differ from thenormal proteomic profile of amniotic fluid in a statisticallysignificant manner. In addition, the Examples present expression markersand unique expression signatures characteristic of IAI and Downsyndrome, respectively.

Statistical methods for comparing proteomic profiles are well known inthe art. For example, in the case of a mass spectrum, the proteomicprofile is defined by the peak amplitude values at key mass/charge (M/Z)positions along the horizontal axis of the spectrum. Accordingly, acharacteristic proteomic profile can, for example, be characterized bythe pattern formed by the combination of spectral amplitudes at givenM/Z vales. The presence or absence of a characteristic expressionsignature, or the substantial identity of two profiles can be determinedby matching the proteomic profile (pattern) of a test sample with theproteomic profile (pattern) of a reference or normal sample, with anappropriate algorithm. A statistical method for analyzing proteomicpatterns is disclosed, for example, in Petricoin III, et al., The Lancet359:572-77 (2002).; Issaq et al., Biochem Biophys Commun 292:587-92(2002); Ball et al., Bioinformatics 18:395-404 (2002); and Li et al.,Clinical Chemistry Journal, 48:1296-1304 (2002).

4. Screening Assays

The proteomic profiles of the invention find further utility inscreening assays to identify drug candidates for the treatment of aparticular maternal/fetal condition. Such screening assays are based onthe ability of a test molecule to convert a proteomic profile containingan expression signature characteristic of the maternal/fetal conditionto be treated into a proteomic profile devoid of the expressionsignature. In one particular embodiment, the ability of the testcompound to convert a pathologic expression profile into a normalexpression profile is tested. In another embodiment, the screening assaytests the ability of a test compound to convert a unique expressionsignature characteristic of a pathologic condition into a correspondingnormal expression signature.

Such screening assays can be performed in vitro by treatment of adiseased biological sample and comparing the proteomics expressionprofiles before and after treatment. Alternatively or in addition, drugscreening can be performed by treating a laboratory animal exhibitingthe target pathologic maternal/fetal condition with a test compound,taking samples of a biological fluid of the animal before and aftertreatment, and comparing the proteomic profiles of the two samples. Inthis assay, it is also possible to take samples of biological fluid atvarious time points following treatment, and follow the time course oftreatment. These methodologies may be applied also to characterize thetoxicology of pharmaceutical agents, as well as to identify optimalcandidates for specific therapies.

The test compounds can, for example, be peptides, non-peptide smallorganic molecules, proteins, polypeptides, antibodies (includingantibody fragments), antisense molecules, oligonucleotide decoys, andany other classes of molecules that have been used previously as drugsor drug candidates.

The biological fluid can, for example, be amniotic fluid, serum (e.g.maternal serum), plasma, urine, cerebrospinal fluid, breast milk, mucus,or saliva.

Therapeutically active compounds identified can be formulated inconventional pharmaceutical formulations. A compendium of art-knownformulations is found in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Company, Easton, Pa. Reference to this manualis routine in the art.

5. Protein Arrays

Both the diagnostic and the screening assays discussed above can beperformed using protein arrays. In recent years, protein arrays havegained wide recognition as a powerful means to detect proteins, monitortheir expression levels, and investigate protein interactions andfunctions. They enable high-throughput protein analysis, when largenumbers of determinations can be performed simultaneously, usingautomated means. In the microarray or chip format, that was originallydeveloped for DNA arrays, such determinations can be carried out withminimum use of materials while generating large amounts of data.

Although proteome analysis by 2D gel electrophoresis and massspectrometry, as described above, is very effective, it does not alwaysprovide the needed high sensitivity and this might miss many proteinsthat are expressed at low abundance. Protein microarrays, in addition totheir high efficiency, provide improved sensitivity.

Protein arrays are formed by immobilizing proteins on a solid surface,such as glass, silicon, micro-wells, nitrocellulose, PVDF membranes, andmicrobeads, using a variety of covalent and non-covalent attachmentchemistries well known in the art. The solid support should bechemically stable before and after the coupling procedure, allow goodspot morphology, display minimal nonspecific binding, should notcontribute a background in detection systems, and should be compatiblewith different detection systems.

In general, protein microarrays use the same detection methods commonlyused for the reading of DNA arrays. Similarly, the same instrumentationas used for reading DNA microarrays is applicable to protein arrays.

Thus, capture arrays (e.g. antibody arrays) can be probed withfluorescently labelled proteins from two different sources, such asnormal and diseased biological fluids. In this case, the readout isbased on the change in the fluorescent signal as a reflection of changesin the expression level of a target protein. Alternative readoutsinclude, without limitation, fluorescence resonance energy transfer,surface plasmon resonance, rolling circle DNA amplification, massspectrometry, resonance light scattering, and atomic force microscopy.

For further details, see, for example, Zhou H, et al., TrendsBiotechnol. 19:S34-9 (2001); Zhu et al., Current Opin. Chem. Biol.5:40-45-(2001); Wilson and Nock, Angew Chem Int Ed Engl 42:494-500(2003); and Schweitzer and Kingsmore, Curr Opin Biotechnol 13:14-9(2002). Biomolecule arrays are also disclosed in U.S. Pat. No.6,406,921, issued Jun. 18, 2002, the entire disclosure of which ishereby expressly incorporated by reference.

Further details of the invention will be apparent from the followingnon-limiting examples.

Example 1 General Protocols

Primate Model of Intra-Amniotic Infection

This protocol was approved by the Institutional Animal Care UtilizationCommittee of the Oregon National Primate Research Center, and guidelinesfor humane care were followed. Three pregnant rhesus monkeys (Macacamulatta) with timed gestations were chronically catheterized aspreviously described (Haluska G J, et al., Temporal changes in uterineactivity and prostaglandin response to RU 486 in rhesus macaques in lategestation, Am J Obstet Gynecol 157: 1487-95 (1987); and Gravett M G, etal., An experimental model for intramniotic infection and preterm laborin rhesus monkeys. Am J Obstet Gynecol 171: 1660-7 (1994)). Briefly, atapproximately day 110 of gestation (term is 167 days) pregnant animalswere conditioned to a jacket and tether system (Ducssay C A, et al.,Simplified vest and tether system for maintenance of chronicallycatheterized pregnant rhesus monkeys. Lab. Anim Sci 38:343-4 (1988)).After conditioning, intrauterine surgery was performed between days 119and 126 of gestation under general anesthesia. Maternal femoral arterialand venous catheters, fetal arterial and venous catheters, twoopen-ended intra-amniotic pressure catheters, myometrialelectromyographic electrodes, and fetal electrocardiographic electrodeswere surgically implanted. All animals received terbutaline sulfate (1mg intravenously over 3 to 5 hours twice daily) for 1 to 5 days aftersurgery to control uterine irritability. Animals also received cefazolin(250 mg intravenously every 12 hours), which was discontinued at least48 hours before inoculation of bacteria.

After postoperative stabilization for 8 to 13 days (day 126 to 138 ofgestation), intra-amniotic infection was established by intra-amnioticinoculation of 106 colony-forming units (cfu) of group B Streptococcus,type III, grown in overnight cultures in Todd-Hewitt broth, centrifuged,washed, and suspended in 0.5 ml of saline solution (n=3 animals), 10⁷cfu of Ureaplasma urealyticum (1 animal) or Mycoplasma hominis (1animal), grown in broth. Amniotic fluid samples were collected seriallyfrom all animals during the study period (daily before inoculation andevery 4 to 12 hours after inoculation) for quantitative bacterialcultures, white blood cell analysis by hemocytometer, and cytokine andprostaglandin concentrations (previously reported—Gravett M G, et al.,An experimental model for intra-amniotic infection and preterm labor inrhesus monkeys. Am J Obstet Gynecol 171: 1660-7 (1994)).

Fetal electrocardiographic and uterine activity (electromyographic andintra-amniotic pressure) were continuously recorded from surgery untildelivery. Uterine contractility was recorded as the area under thecontraction curve per hour and expressed as the hourly contraction area(HCA) in millimeters of mercury times seconds/hour.

The maternal cervix was palpated vaginally before infection and seriallythereafter. Consistency, effacement, and dilatation were recorded ateach examination. After delivery, by cesarean section in all except oneanimal and vaginally in one animal, decidual, placental, and intermembrane bacterial cultures were obtained form infected animals toconfirm infection and histopathologic studies were performed.

Amniotic Fluid Assays

Amniotic fluid samples (3 ml) were immediately centrifuged aftercollection at 3,000 rpm and 4° C. for 20 minutes. The sediment was savedfor cellular analysis and the supernatant stored in pyrogen-free sterilevials at −20° C. until assayed.

Human Study

The study population was drawn from 309 women admitted in prematurelabor with intact fetal membranes to the University of WashingtonMedical Center or associated hospitals in Seattle between Jun. 25, 1991and Jun. 30, 1997, as previously described (Hitti J, et al., Amnioticfluid tumor necrosis factor-α and the risk of respiratory distresssyndrome among preterm infants. Am J Obstet Gynecol 177:50-6 (1997)).All women provided written informed consent, and the study protocol wasapproved by the Institutional Review Boards for all participatinghospitals. The participants were at gestational ages of 22 to 34 weeksby last menstrual period or from the earliest available ultrasound. Allparticipants had intact fetal membranes at study enrollment. Pretermlabor was defined as regular uterine contractions at a frequency of 10minutes with either documented cervical change or a cervical dilatationof 1 centimeter or effacement of 50%. Women with cervical dilatation >4centimeters or ruptured membranes at admission were excluded. Women withmultiple gestations, cervical cerclage, placenta previa, abruptioplacentae, diabetes, hypertension, and pre-eclampsia were consideredeligible if they otherwise met study criteria.

Transabdominal amniocentesis was performed under ultrasound guidance forall study participants and maternal venous blood was also collected byvenipuncture at the time of enrollment From this study population, asubset (Tables 1A and B) was retrospectively identified for proteomicanalysis as reported here. This subset included 11 patients withevidence of intrauterine infection (as defined by the recovery of amicrobial pathogen form amniotic fluid or an amniotic fluid IL-6concentration of ≧2,000 pg/ml), and a randomly selected subset of 11patients without intrauterine infection but with preterm birth and 11patients without infection and with preterm labor responsive totocolytic therapy and who had subsequent term birth. These patientsconstitute the study population for this report.

The study population was divided into three groups: 1) those patientswith evidence of intrauterine infection, based upon either recovery ofmicroorganisms from amniotic fluid or an amniotic fluid IL-6concentration of >2,000 pg/ml; 2) those patients with preterm labor anddelivery prior to 35 weeks of gestation without evidence of intrauterineinfection; and 3) those patients with preterm labor responsive totocolytic therapy who delivered at ≧35 weeks of gestation. There were nodifferences in maternal age, race, or parity between these three groups(Tables 1A and B). However, patients with intrauterine infection wereseen at a somewhat earlier gestational age at enrollment (p=0.10) anddelivered at a significantly earlier gestation age than those patientswith preterm delivery without infection or those with term delivery(27.3±0.9 weeks versus 29.8±1.0 and 37.0±0.9 weeks respectively,p<0.0001). In addition, those with intrauterine infection had asignificantly shorter enrollment to delivery interval (2.1±5.6 days,compared to 8.4±6.3 and 46.9±5.6 days for the other two groups,p<0.0001). Ninety-one percent of those with intrauterine infectiondelivered within seven days of enrollment.

Among those eleven patients with infection, microorganisms wererecovered from four (2 with Escherichia coli, 1 with Candida albicans,and 1 with mixed anaerobes); all of these patients delivered withinseven days. Seven other patients were identified based upon amnioticfluid IL-6 concentrations of greater than 2,000 pg/ml. The mean amnioticfluid concentration of interleukin-6 was 27.7±7.8 ng/ml among thesepatients, compared to 0.68±0.20 ng/ml among those with preterm deliverywithout infection and 0.25±0.13 ng/ml among those with preterm labor andterm delivery (p<0.01).

TABLE 1A Characteristics of the Study Population Group GROUP 3 PML withGROUP 1 subsequent PMD GROUP 2 term GROUP with IUI PMD without IUIdelivery 3 vs 1 Characteristic (n = 11) (n = 11) (n = 11) p valueMaternal Age 24.5 ± 5.4 26.6 ± 9.0 25.6 ± 6.0 NS White Race 6 (55%) 4(36%) 6 (55%) NS Parity  1.9 ± 1.6  1.9 ± 1.5  3.0 ± 2.5 NS Nulliparity3 (27%) 1 (9%)  1 (9%)  NS Gestational Age 26.9 ± 1.1 28.6 ± 1.1 30.3 ±1.1 0.10 at Enrollment (wks) Gestational Age 27.3 ± 0.9 29.8 ± 1.0 37.0± 0.9 <0.0001 at Delivery (wks) Enrollment to  2.1 ± 5.6  8.4 ± 6.3 46.9± 5.6 <0.0001 Delivery Interval (days) Delivery. 7 days 10 (91%)  6(55%) 0 <0.001

Data expressed as mean standard deviation. Analysis by ANOVA forcontinuous data and Chi-square for categorical data. Abbreviations: PMD,premature delivery <35 weeks; IUI, intrauterine infection; PML,premature labor without delivery.

TABLE 1B Screening Results. Group GROUP 3 PML with subsequent termdelivery GROUP 1 GROUP 2 (n = 11) PMD with IUI PMD without IUI p valueLCharacteristic (n = 11) (n = 11) GROUPS 3 VS 1 Bacterial culture 4/110/11 0/11 p < 0.01 positive IL-6 positive 7/11 0/11 0/11 p < 0.01Diagnostic 11/11   2/11* 0/11 p < 0.01 protein profiles *The twopositive samples in this pool represent subclinical infection since theAF of the two subjects demonstrated low levels of bacteria, positive byPCR procedures. This indicates that protein profiling can be used toidentify subclinical intraamniotic infection.

In the foregoing tables, data are expressed as mean standard deviation.Analysis by ANOVA for continuous data and Chi-square for categoricaldata. Abbreviations: PMD, premature delivery <35 weeks; IUI,intrauterine infection; PML, premature labor without delivery.

TABLE 1C Fisher's test significance values for screening test results.Fisher's Exact: PMD with IUI vs PML p < 0.05 (one-sided) BacterialCulture Class Positive Negative Total PMD with IUI 4 7 11 PML, termdelivery 0 11 11 Total 4 18 22 Fisher's Exact: PMD with IUI vs PML p <0.01 (one-sided) IL-6 Status Class Positive Negative Total PMD with IUI7 4 11 PML, term delivery 0 11 11 Total 7 15 22 Fisher's Exact: PMD withIUI vs PML p < 0.005 (one-sided) Diagnostic Protein Profile ClassPositive Negative Total PMD with IUI 11 0 11 PML, term delivery 0 11 11Total 11 11 22 Fisher's Exact: PMD with IUI vs PMD without IUI p < 0.005(one-sided) Diagnostic Protein Profile Class Positive Negative Total PMDwith IUI 11 0 11 PMD without IUI 2 9 11 Total 13 9 22 Fisher's Exact:PMD without IUI vs PML p n.s. Diagnostic Protein Profile Class PositiveNegative Total PMD without IUI 2 9 11 PML, term delivery 0 11 11 Total 220 22

Proteomic Analysis of Amniotic Fluid

1-Dimensional (1-D) Gel Electrophoresis

100 μg of amniotic fluid after reduction with iodoacetamide was loadedon a 15% SDS-PAGE gel. Electrophoresis was conducted at 80V to separatethe proteins in the sample. After electrophoresis the gel was stainedwith Coomasie blue R-250 and images were collected using Bio-Rad GS800densitometer and PDQUEST software. Individual bands were cut from thegel, destained and digested in-gel with trypsin for 24-48 hrs at 37° C.The peptides were extracted with 0.1% TFA and dried in a speedvac. Theextract was dissolved in 0.1% TFA and purified using Zip Tip_(c18)pipette tips from Millipore. (Marvin L., et al. Identification ofproteins from one-dimensional sodium dodecyl sulfate-polyacrylamide gelelectrophoresis using electrospray quadrupole-time-of-flight tandem massspectrometry. Rapid Commun Mass Spectrom. 14(14):1287-92, 2000).

2-Dimensional (2-D) Gel Electrophoresis

Amniotic fluid (400-2000 μg) with or without removal of albumin wasdissolved in IEF buffer and rehydrated on to a 24 cm IPG strip (pH 3-10)for 12 h at room temperature. After rehydration, the IPG strip wassubjected to 1-dimension electrophoresis at 70˜90 kVhrs. The IPG stripwas then equilibrated with DTT equilibration buffer I and IAAequilibration buffer II for 15 minutes sequentially, before seconddimension SDS-PAGE analysis. The IPG strip was then loaded on to a 4˜20%SDS-PAGE gel and electrophoresis conducted at 120 V for 12 hrs toresolve proteins in the second dimension. The gel was stained withCoomassie Blue R-250 and imaged using Bio-Rad GS800 densitometer andPDQUEST software. Individual spots were cut from the gel, destained anddigested in-gel with trypsin for 24-48 hrs at 37 C. The peptides wereextracted with 0.1% TFA and purified using Zip Tip_(c18) pipette tipsfrom Millipore (2-D Proteome analysis protocols: Methods in MolecularBiology: 112, 1999).

HPLC Fractionation

Human amniotic fluid samples after removal of albumin and IgG (1-15 mgprotein) were dissolved in 20 mM Tris-HCl, pH 7.5. Anion-exchangechromatography was performed using TSK gel DEAE-5PW column on a Waters1525 HPLC equipped with an auto sampler and a UV absorbance detector. Alinear salt elution gradient was used to fractionate the proteins.Fractions were collected at one minute intervals. Fractions were pooled,digested with trypsin and peptide mixtures were analyzed using the massspectrometer (Q-Tof-2).

Mass Spectrometry Analysis

(1) Q-Tof-2

Samples after in-gel digestion were analyzed on a Micromass Q-Tof-2 massspectrometer connected to a Micromass CapLC. The Q-Tof-2 was equippedwith a regular Z-spray or nanospray source and connected to aIntegrafrit C18 75 um ID×15 cm fused silica capillary column. Theinstrument was controlled by, and data were acquired on, a Compaqworkstation with Windows NT and MassLynx 3.5 software. The Q-Tof-2 wascalibrated using Glu1Fibrinopeptide B by direct infusion or injectioninto the CapLC. A MS/MSMS survey method was used to acquire MS/MSMSspectra. Masses 400 to 1500 were scanned for MS survey and masses 50 to1900 for MSMS. Primary data analysis was performed on a PC with Windows2000 and SEQUEST (version 1.3) and/or LUTEFISK. Peak lists weregenerated, using the built-in automatic functions for peak-picking andapplying centroid-fitting to each peak.

(2) LCQ-MS

Protein spots from dried Coomassie blue stained gels were excised andrehydrated/washed for 30 min. in 0.5 ml of 20 mM ammonium bicarbonate,50% acetonitrile solution. The gel regions were then dried by vacuumcentrifugation and digested insitu by rehydrating in 20 nM sequencinggrade modified trypsin (ProMega, Madison, Wis., USA) using the method ofCourchesne and Patterson, Identification of proteins by matrix-assistedlaser desorption/ionization masses, Methods Mol. Biol. 112:487-511(1999). Tryptic digests were then concentrated by vacuum centrifugation,separated by reverse phase chromatography, and peptides analyzed by amodel LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, Calif.).Samples were separated with Zorbax C-18 0.5 mm×150 mm microbore columnusing a 10 μL min⁻¹ flow rate and a gradient of 0 to 40% B (75%Acetonitrile in water) over one hour with an 1100 Capillary LC System(Agilent Technologies, Foster City, Calif.). Peptides were introduceddirectly into the standard ThermoFinnigan electrospray source. MS/MSspectra were acquired in an automated fashion using standard LCQsoftware and then analyzed further using SEQUEST (ThermoFinnigan). Forfurther details see, Courchesne, P. L. and Patterson, S. D., supra.

Data Analysis

(1) Sequest and DTASelect

Automated analysis of tandem mass spectra (MS/MS) was performed usingSEQUEST software (ThermoFinnigan) as described by Yates et al., MethodsMol. Biol. 112:553-69 (1999). SEQUEST matches uninterrupted tandem massspectra to database peptide sequences. Searches were run with thedefault parameters using a combined indexed non-redundant database ofprotein sequences obtained from the Protein Information Resource(release date) and SwissProt (release date). The database wasconstructed using the Xcalibur Database Manager (ThermoFinnigan).S-Carboxyamidated cysteine was the only considered modification.

Sequest results were further analyzed using DTASelect (The ScrippsResearch Institute, Tabb, 2002). DTASelect organizes and filters SEQUESTidentifications. The default parameters were used except as follows: 1)any database matches including the string “keratin” in the proteindescription were excluded and 2) spectra from the LCQ mass spectrometerwere filtered with a cross correlation score cut-off of 2.4 for thedoubly charged ions. Each spectra and proposed sequence pair selected byDTASelect were visually inspected and the final results were input intoa spreadsheet (Microsoft Excel) or a database (Microsoft Access) formanagement.

For further details, see also: Tabb D L, et al., DTASelect and Contrast:Tools for Assembling and Comparing Protein Identifications from ShotgunProteomics. J. Proteome Res. 1:21-26 (2002).

(2) Lutefisk

Automated de novo sequencing of all spectra was performed using acomputer program, Lutefisk 1900 v1.2.5 (Taylor J A, Johnson R S.Implementation and uses of automated de novo peptide sequencing bytandem mass spectrometry. Anal Chem 73(11):2594-604 (2001). Lutefiskgenerates peptide sequences for spectra of which some are sufficientlydetailed for homology-based sequence searches. Modifications,acrylamide, carbamidomethylation, and phosphorylation, were considered.

MALDI Detection Protocol and Parameters

MALDI mass spectrometry was performed on a custom-built time-of-flightreflector mass spectrometer (Jensen O N, et al., Direct observation ofUV-crosslinked protein-nucleic acid complexes by matrix-assisted laserdesorption ionization mass spectrometry. Rapid Commun Mass Spectrom7(6):496-501 (1993)) equipped with a two-stage delayed extractionsource. Approximately 1 μL of sample solution was mixed with 1 μL SA(Sinapinic acid in 60:40 water/acetonitrile 0.1% TFA final conc.) A 1.0μL droplet of this analyte/matrix solution was deposited onto a matrixpre-crystallized sample probe and allowed to dry in air. Mass spectrawere produced by radiating the sample with a (355 nm) Nd:YAG laser(Spectra Physics) and operating the ion source at 23 kV with a 700ns/1.0 kV delay. Every mass spectrum was recorded as the sum of 20consecutive spectra, each produced by a single pulse of photons. Ionsfrom an added standard were used for mass calibration.

SELDI Analysis of Amniotic Fluid

A total of 0.5-3.0 ug protein from amniotic fluid samples was spotted ona Normal Phase NP20 (SiO₂ surface), Reverse Phase H4 (hydrophobicsurface: C-16 (long chain aliphatic), or immobilized nickel (IMAC) SELDIProteinChip® array (Ciphergen Biosystems, Inc. Fremont, Calif.). Afterincubation at room temperature for 1 hour, NP1 and H4 chips weresubjected to a 5 ul water wash to remove unbound proteins andinterfering substances (ie buffers, salts, detergents). After air-dryingfor 2-3 minutes, two 0.5 ul applications of a saturated solution ofsinapinic acid in 50% acetonitrile (v/v), 0.5% trifluoroacetic acid(v/v) was added and mass analysis was performed by time-of-flight massspectrometry in a Ciphergen Protein Biology System II (PBS II), Issaq,J. H, et al.: The SELDI-TOF MS Approach to proteomics: Protein Profilingand Biomarker Identification. Biochem Biophys Res Commun. 5;292(3):587-92, 2000.

Example 2 Identification of Proteins and Polypeptides Expressed in theAmniotic Fluid

Using the materials and methods described in Example 1, proteins andpolypeptides expressed in normal and infected amniotic fluid wereidentified. Human and primate amniotic fluid samples (pooled andindividual) were subjected to protein separation techniques (1-D, 2-Dand HPLC fractionation) as described in Example 1. The separatedproteins (gel bands, spots and fractions) were digested with trypsin togenerate peptide pools. The peptide pools were analyzed using tandem MSto decipher their amino acid sequence and composition.

Five thousand MS spectra were selected using spectral verificationprograms. These spectral files were analyzed using de novo sequencingprograms (Lutefisk, Peaks) to generate the amino acid sequencecorresponding to each peptide. The de novo sequences generated from thepeptide pool were used to search protein and DNA databases as describedin Example 1.

Using homology maps and sequence verification, expression of a varietyof proteins was discovered in the amniotic fluid. The detected proteinswere analyzed for potential function based on known structuralsimilarities (sequence homology maps). Proteins belonging to importantfunctional classes involved in a wide range of diseases were discovered.Proteins and polypeptides discovered for the first time in the humanamniotic fluid are listed in the following Table 2 under these potentialfunctional categories.

Proteins shown to be differentially expressed by immunoassays also, andproteins more abundantly or uniquely represented in the infectedamniotic fluid are separately marked. In this context, relativeabundance is defined as the amount of the peptides representing acertain polypeptide or protein in a test sample, relative to a referencesample. Accordingly, a protein is more abundantly represented ininfected amniotic fluid if more peptides derived from the same proteinare present in infected amniotic fluid than in a non-infected referencesample of amniotic fluid.

Table 3 lists proteins and polypeptides previously known to be presentin amniotic fluid, the presence of which was reaffirmed by the presentassays. Proteins which are known markers for infection related eventsare separately marked.

TABLE 2 Proteins and polypeptides discovered for the first time in thehuman amniotic fluid GenBank Acc. No Protein ID Protein Name Immuneresponse related genes U12026 CAPG_HUMAN Macrophage capping protein#X83006 NGAL_HUMAN Neutrophil gelatinase-associated lipocalin# M19507PERM_HUMAN Myeloperoxidase precursor# M22300 PLSL_HUMAN L-plastin(Lymphocyte cytosolic protein 1)* NM001700 AZU1_HUMAN Azurocidin# Z38026FA39_HUMAN Antibacterial protein FALL-39 precursor# AF159456 Q9UKJ4Gp-340 variant protein AL355392 Q9H4V6 Novel protein similar to mousevon Ebner salivary gland protein, isoform 2 M93056 ILEU_HUMAN Leukocyteelastase inhibitor# Y00278 S108_HUMAN Calgranulin A*# X06233 S109_HUMANCalgranulin B Structural proteins D00682 COF1_HUMAN Cofilin, non-muscleisoform M69066 MOES_HUMAN Moesin (Membrane-organizing extension spikeprotein) J03191 PRO1_HUMAN Profilin I*# D44497 CO1A_HUMAN Coronin-likeprotein p57 (Coronin 1A) D00017 ANX2_HUMAN Annexin II (Lipocortin II)M15801 FINC_HUMAN Fibronectin precursor M17783 GDN_HUMAN Glia derivednexin precursor# Proteases and protease inhibitors M21642 ANT3_HUMANAntithrombin-III precursor S66896 SCC1_HUMAN Squamous cell carcinomaantigen 1 U19576 SCC2_HUMAN Squamous cell carcinoma antigen 2 AB006423SPI2_HUMAN Serpin I2 precursor# X05978 CYTA_HUMAN Cystatin A (Stefin A)(Cystatin AS)# U46692 CYTB_HUMAN Cystatin B (Liver thiol proteinaseinhibitor) X05607 CYTC_HUMAN Cystatin C precursor Transporters andbinding proteins Y00856 IBP1_HUMAN Insulin-like growth factor bindingprotein 1- Proteolytic fragment (only)* L10641 VTDB_HUMAN VitaminD-binding protein precursor J00098 APA1_HUMAN Apolipoprotein A-Iprecursor (Apo-AI) X57348 143S_HUMAN 14-3-3 protein sigma (Stratifin)M86400 143Z_HUMAN 14-3-3 protein zeta/delta X04412 GELS_HUMAN Gelsolinprecursor, plasma X53961 TRFL_HUMAN Lactotransferrin precursor(Lactoferrin) Enzymes and other molecules V00572 PGK1_HUMANPhosphoglycerate kinase 1 J04173 PMG1_HUMAN Phosphoglycerate mutase 1X67688 TKT_HUMAN Transketolase *Proteins shown to be differentiallyexpressed by immunoassays also. #Peptides representing these proteinsare more abundantly or uniquely detected in the infected amniotic fluid.

TABLE 3 Proteins and polypeptides, previously known to be present in theamniotic fluid, identified using de novo sequencing. GenBank Acc. NoProtein ID Protein Name KO2765 CO3_HUMAN Complement C3 precursor* J00241KAC_HUMAN Ig kappa chain C region J00253 LAC_HUMAN Ig lambda chain Cregions J00228 GC1_HUMAN Ig gamma-1 chain C region X57127 H2BF_HUMANHistone H2B.f* X00038 H4_HUMAN Histone H4* J00153 HBA_HUMAN Hemoglobinalpha chain U01317 HBB_HUMAN Hemoglobin beta chain U01317 HBD_HUMANHemoglobin delta chain M91036 HBG_HUMAN Hemoglobin gamma-A and gamma-Gchains Z83742 H2AC_HUMAN Histone H2A M22919 MLEN_HUMAN Myosin lightchain alkali, non-muscle isoform J05070 MM09_HUMAN type IV collagenaseprecursor* V00496 A1AT_HUMAN Alpha-1-antitrypsin precursor* K01500AACT_HUMAN Alpha-1-antichymotrypsin precursor* M12530 TRFE_HUMANSerotransferrin precursor M11714 TTHY_HUMAN Transthyretin precursor(Prealbumin) M13699 CERU_HUMAN Ceruloplasmin precursor* X02544A1AH_HUMAN Alpha-1-acid glycoprotein 2 precursor* X06675 A1AG_HUMANAlpha-1-acid glycoprotein 1 precursor* M12523 ALBU_HUMAN Serum albuminprecursor J00098 APA1_HUMAN Apolipoprotein A-I precursor (Apo-AI) *Knownmarkers for infection related events.

Diagnostic Markers for Intrauterine Conditions:

In view of their known biological functions, several proteins listed inthe foregoing tables are promising candidates for detecting andmonitoring intrauterine conditions. A few examples of such conditionsand the corresponding protein markers are discussed below in greaterdetail.

Actin-Modulating and Related Proteins as Markers of DevelopmentalDefects:

Moesin (Membrane-organizing extension spike protein), listed among thestructural proteins in Table 2, is known to be responsible for linkingtransmembrane proteins to the actin cytoskeleton and implicated invarious cell signaling pathways (Speck O, et al.: Moesin functionsantagonistically to the Rho pathway to maintain epithelial integrity.Nature 2; 421(6918):83-7, 2003). It was shown that Rho-family GTPasesand their effectors to modulate the activities of actin modifyingmolecules such as Cofilin and Profilin (also listed as a structuralprotein in Table 2), resulting in cytoskeletal changes associated withgrowth cone extension or retraction (Tang B L. Inhibitors of neuronalregeneration: mediators and signaling mechanisms. Neurochem Int,42(3):189-203, 2003). Coronin-like protein p57 (yet another structuralprotein listed in Table 2) is also involved in actin cross-linking andcapping (Weitzdoerfer R et al.: Reduction of actin-related proteincomplex ⅔ in fetal Down syndrome. Biochem Biophys res Commun. 293:836,2002) and are dysregulated in known developmental defects. Gelsolin(see, the Gelsolin precursor listed an a transporter/binding protein inTable 2), another actin-modulating protein is also known to bedevelopmentally regulated and important in organ systems (Arai M,Kwiatkowski D J. Differential developmentally regulated expression ofgelsolin family members in the mouse. Dev Dyn, 215, 297, 1999). 14-3-3proteins are also known epithelial markers which participate in signaltransduction and differentiation pathways and are essential for normaldevelopment of brain and other vital organs (Wu C, Muslin A J. Role of14-3-3 proteins in early Xenopus development. Mech Dev, 119, 45, 2002).

Accordingly, the listed actin-modulating proteins and other relatedmolecules with important roles during development, that were identifiedfor the first time in human amniotic fluid, could be used to detectdevelopmental defects of various organ systems such as, central nervoussystem, cardiovascular system and other musculoskeletal deformities,which can, for example, result from chromosomal aneuploides. This isparticularly true for Profiling I, which has been shown to bedifferentially expressed in infected amniotic fluid, and thedifferential expression of which has been confirmed by immunoassay.

Markers of Infection and Immune-Response Related Disorders:

The present detection of macrophage capping protein, leukocyte elastase,neutrophil gelatinase-associated lipocalicn, myleoperoxidase, L-plastin(lymphocyte cytosolic protein) and calgranulins (see the list of immuneresponse related genes in Table 2) infected amniotic fluid is the firstdemonstration of the presence and regulation of these proteins inintraamniotic infection. Several of these proteins are known respondersof immune cells in response to infection, inflammation and stress.Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein whichmodulates actin filaments and involved in inflammatory process (Dabiri GA, Molecular cloning of human macrophage capping protein cDNA. A uniquemember of the gelsolin/villin family expressed primarily in macrophagesJ Biol Chem 15; 267(23):16545-52, 1992). Similarly, Calgranulins arecalcium binding proteins known to play a role in injury and woundhealing (Thorey I S. et al. The Ca2+-binding proteins S100A8 and S100A9are encoded by novel injury-regulated genes. J Biol Chem 21;276(38):35818-25, 2001). Leukocyte elastase and neutrophilgelatinase-associated lipocalcin (NGAL) are involved in bacteriostaticand baceterolysis mechanisms (Goetz D H. et al. The neutrophil lipocalinNGAL is a bacteriostatic agent that interferes with siderophore-mediatediron acquisition: Mol Cell 10(5):1033-43, 2002).

In addition to the above immunomodulators we also discovered, for thefirst time, two antibacterial proteins Fall-39 and azurocidin in theinfected amniotic fluid. Antibacterial protein Fall-39 (LL-37) binds tobacterial lipopolysaccharides (ips), and is expressed in bone marrow,testis and neutrophils. Fall-39 stimulates the degranulation of mastcells, and is a potent chemotactic factor for mast cells. Besides itsantibacterial activities, Fall-39 may have the potential to recruit mastcells to inflammation foci. In the presence of the basal medium E,synthetic FALL-39 was highly active against Escherichia coli D21 andBacillus megaterium Bm11. A protective role for Fall 39 has beenproposed, when the integrity of the skin barrier is damaged,participating in the first line of defense, and preventing localinfection and systemic invasion of microbes (Agerberth B, et al.:FALL-39, a putative human peptide antibiotic, is cysteine-free andexpressed in bone marrow and testis. Proc Natl Acad Sci USA, 3;92(1):195-9, 1995).

Azurocidin (CAP37) is a cationic antimicrobial protein isolated fromhuman neutrophils and has important implications in host defense andinflammation. It is released during inflammation and regulatesmonocyte/macrophage functions, such as chemotaxis, increased survival,and differentiation (Pereira H A. CAP37, a neutrophil-derivedmultifunctional inflammatory mediator. J Leukoc Biol; 57(6):805-12,1995).

Proteases and protease inhibitors play a key role in protein regulationand thus control several key physiological mechanisms. We haveidentified the expression of Serpin family of proteases (Serpin,squamous cell carcinoma antigen 1 & 2, glia derived nexin) for the firsttime in human amniotic fluid, including intraamniotic infection. Theserpin superfamily of serine proteinase inhibitors has a central role incontrolling proteinases in many biological pathways and implicated inconformational diseases, such as the amyloidoses, the prionencephalopathies and Huntington and Alzheimer disease (Lomas D A,Carrell R W, Serpinopathies and the conformational dementias. Nat RevGenet; 3:759, 2002).

Additionally, in intraamniotic infection we identified the expression ofCystatins, well known proteinase inhibitors involved in immunomodulation(Vray B, Hartmann S, Hoebeke J. Immunomodulatory properties ofcystatins. Cell Mol Life Sci ;59(9):1503-12, 2002).

The listed proteins are promising markers of infection and/orimmune-response related disorders.

It is noteworthy that peptides representing macrophage capping protein,neutrophil gelatinase-associated lipocalin, myeloperoxidase precursor,L-plastin, azurocidin, antibacterial protein Fall-39, calgranulin A,profilin I, glia-derived nexin, serpin 12, and cystatin A were moreabundantly or uniquely detected in infected amniotic fluid relative tonormal amniotic fluid, and/or showed differential expression inimmunoassays. Accordingly, these proteins are particularly important asmarkers of intra-amniotic infection and/or immune-response relateddisorders.

Other Disease (Infection) Specific Proteins Detected in Human AmnioticFluid

Gp-340 variant protein listed in Table 2, which has been detected inhuman infected amniotic fluid, is a scavenger receptor previouslyidentified in lung. This protein is known to bind to bacteria(streptococcus and variants) The detection of this protein in infectedamniotic fluid complements the sensitive proteomic approach of thepresent invention to identify biomarkers for IAI. Thus, Gp-340 variantprotein identified in the infected amniotic fluid lends itself for thedetection of neonatal sepsis).

IGFBP-1 (Proteolytic Fragment)

As shown in Table 2, IGFBP-1 has been shown to be differentiallyexpressed in infected amniotic fluid. The insulin-like growth factor(IGF) systems is critically involved in fetal and placental growth andmodulates steroid hormone actions in the endometrium throughautocrine/paracrine mechanisms. IGF-I and IGF-II stimulatedproliferation and differentiation, and maintain differentiated cellfunctions in several cell types in vitro. Endometrial stromal cellsproduce IGF-I and IGF-II as well as the high affinity IGF-bindingproteins (IGFBPs). The mRNA of six high affinity IGFBPs, which canmodulate IGF actions, are expressed in human endometrium. The mostabundant IGFBP in human endometrium is IGFBP-1, which is secreted bypredecidualized/decidualized endometrial stromal cells in late secretoryphase and during pregnancy. This has implications for clinicalobstetrics and gynecology, where there is evidence for apathophysiological role for IGFBP-1 in pre-eclampsia, intrauterinegrowth restriction, polycystic ovarian syndrome and trophoblast andendometrial neoplasms.

The presence and regulation of an IGFBP-1 proteolytic fragment in humanamniotic fluid and maternal serum opens a new way for monitoringintrauterine and maternal conditions associated with pregnancy.

For further details see, also Example 12 below.

Example 3 Protein Expression Profiles of Primate Amniotic FluidFollowing Intrauterine Infection

Protein expression profiles of primate amniotic fluid followingintrauterine infection, compared with the corresponding normalexpression profiles, are shown in FIGS. 1A-C.

As illustrated in FIGS. 1A-C, the global protein expression profiles ofcontrol and infected amniotic fluid are distinct. A detailed spectra ofamniotic fluid profiles in a smaller mass range (FIGS. 1B and 1C), showsdistinct and characteristic differences between the protein expressionprofiles of control and infected samples approximately in the 3-5 KDaand 10-12 KDa range. This illustrates global regulation of proteinexpression in response to intrauterine infection and the ability todetect a unique expression signature diagnostic of intrauterineinfection.

Example 4 Early Detection of Diagnostic Pattern/Profile of Infection inthe Primate Amniotic Fluid

FIG. 2 shows the time course analyses of the primate amniotic fluid inresponse to infection (GBS). Amniotic fluid was collected before theinoculation of bacteria and serially after infection and subjected toSELDI-TOF analysis as described in Example 1. FIG. 2A shows the proteinexpression profile before infection, FIG. 2B 12 hours after infection,and FIG. 2C 36 hours after infection.

As shown in FIG. 2C, one of the diagnostic peaks (10-11 KDa) ofintrauterine infections clearly reaches high levels of expression within36 hours of acute infection. This demonstrates that diagnostic proteinprofiles can be used for monitoring the disease state and response totreatment.

Example 5 Protein Expression Profiles of Human Amniotic Fluid FollowingIntrauterine Infection

FIG. 3 shows the results of SELDI-TOF analysis of amniotic fluidextracts bound to chemically defined normal phase chip arrays. FIG. 3Ashows the whole spectrum at 235 laser intensity. FIG. 3B is a detailedspectrum showing the differences between infected and control samples inthe 10-12 kDa region. FIG. 3 is a detailed spectrum showing thecharacteristic differences between infected and control samples in the3-5 kDa region.

As shown in FIGS. 3A-D, the global protein expression profiles ofcontrol and infected amniotic fluid are distinct. A detailed spectra ofamniotic fluid profiles in a smaller mass range (FIGS. 3B and C), showsdistinct over expressed proteins (3-5 KDa and 10-12 KDa range) betweencontrol and infected samples. Analysis of protein peaks relativeintensities, suggests the presence of two distinct diagnostic clusters(10-12 kDa and 3-5 kDa ranges). This illustrates global regulation ofprotein expression in response to intrauterine infection and the abilityto detect a unique expression signature diagnostic of intrauterineinfection both in human and primate models.

It is noteworthy that the diagnostic pattern of human amniotic fluid isin good agreement with the diagnostic pattern of primate amniotic fluid(Examples 3 and 4).

Example 6 Generation of Diagnostic Profiles Using Different MassSpectrometers

The diagnostic protein expression profile can be detected usingdifferent types of mass spectrometers. It has been examined whetherdifferent mass spectrometers produce similar diagnostic profiles. If thediagnostic profiles are substantially independent on the type of massspectrometer, the detected differential protein expression in theamniotic fluid can provide a diagnostic signature for intrauterineinfection.

FIG. 4 shows mass spectra acquired on a generic MALDI-TOF massspectrometer (Jensen O N, et al., Direct observation of UV-crosslinkedprotein-nucleic acid complexes by matrix-assisted laser desorptionionization mass spectrometry. Rapid Commun Mass Spectrom 7(6):496-501(1993)) using amniotic fluid from human control (A), withoutintrauterine infection, and a sample (B) with intrauterine infection.

As shown in FIGS. 4A and B, the diagnostic profile of intrauterineinfection in the 10-12 KDa range is detected using the alternate massspectrometer is similar to the profile detected using the SELDI-TOFmachine. This indicates that differential protein expression profilesare robust and can be detected using a wide range of current massspectrometers.

In summary, it has been discovered that amniotic fluid proteins andpolypeptides exhibit differential expression patterns diagnostic ofdisease state. The results presented here demonstrate thatdisease-specific diagnostic patterns can be detected using multiple massspectrometry approaches. The patterns or protein expression profiles arecomparable between humans and primates. The profiles can be used tomonitor a time-course (infection or treatment) effect.

Example 7 Quantification of Protein and Polypeptide Expression inAmniotic Fluid for Diagnostic and Prognostic Monitoring

SDS-PAGE:

Proteins from human amniotic fluid (AF) containing high salt wasprecipitated with acetone. 100 μg of amniotic fluid proteins was run ona 15% SDS-PAGE. The gel was stained with Coomassie Blue R-250. The gelimage was scanned by Bio-Rad gel Scanner.

FIG. 5 shows the SDS-Coommassie Blue stained gel of A) 4 human controlAF samples pooled; B) individual control AF sample; C) 4 human infectedAF samples pooled; and D) individual infected AF sample.

FIG. 5 shows significant differences between the control and infectedprotein expression levels in the 10-15 KDa range. It has been concludedthat some of the proteins and proteolytic fragments in this massdetected using the mass spectrometers are responsible for the diagnosticprofiles reflective of the protein expression levels, and havediagnostic and prognostic utility.

Example 8 Western Blot Analysis of Amniotic Fluid from IntrauterineInfection

100 μg of AF proteins were run on 4-20% SDS-PAGE at 200 V for 60 minutesand transferred to PVDF membrane at 90 mM for 75 minutes. The membranewas blocked with 5% milk PBST for 45 min at RT and incubated with 1μg/ml primary antibody (Santa Cruz and Dako) overnight at 4 C. Afterwash with TBST 3 times, the membrane was incubated with secondaryantibody IgG-HRP (Sigma) for 90 min at RT and visualized with ECL(Pierce).

The results are shown in FIG. 6: A) Control AF sample (pooled); B)Infected AF sample (pooled). FIG. 6 shows that IGFBP1 (11 KDa), profilinand ceruloplasmin (130 KDa) are expressed at a higher level in infectedAF compared to non-infected AF. L-Plastin levels were lower in theinfected sample compared to control AF sample. These proteins were alsoidentified from the human infected samples using MS approaches (de novosequencing) and are listed in Example 2 above.

Example 9 Immunoprecipitation Analysis of Amniotic Fluid fromIntrauterine Infection

Two micrograms of primary antibody was mixed with 600 μg of AF proteinand incubated at 4° C. overnight. 15 μl of protein G Sepharose beads wasadded and incubated on a shaker for 60 minutes at room temperature. Thebeads were washed with IP buffer for 6 times.

The results are shown in FIG. 7, where (A) shows the control amnioticfluid sample (pooled), and (B) shows the infected amniotic fluid sample.FIG. 7 shows that ceruloplasmin (˜130 KDa) and calgranulin (˜16 KDa) areexpressed at a higher level in the infected amniotic fluid than controlamniotic fluid.

Example 10 Detection of Differential Protein Expression in the HumanAmniotic Fluid and Maternal Serum

It has been examined if the differentially expressed proteins in theamniotic fluid can be used as a lead to measure similar proteins in thematernal serum. This will enable to develop rapid and non-invasivetesting for diagnoses and monitoring. The results are shown in FIG. 8,where (A) is the control sample (pooled), and (B) is the infected sample(pooled). FIG. 8 shows that an IGFBP-1 smaller proteolytic fragment isconsistently differentially expressed both in AF and maternal serum inresponse to intrauterine infection.

Example 11 Protein Microarray Analysis of Amniotic Fluid fromIntrauterine Infection

Antibodies: IGFBP-1 (DSL); complement C3, Desmin, neutrophil elastase,NSE antibody (DAKO); calgranulin, ceruloplasmin, TIMP-1, plastin andprofiling (Santa Cruz).

Antibody spotting: antibodies were dissolved in 40% glycerol, 60% PBS,pH 7.5 at a concentration of 100 μg/ml and were spotted on aldehydeslides using a Arrayer (Cartesian).

Following a 3 hr incubation in a humid chamber at room temperature, theslides were incubated for one hour in a solution of PBS, pH 7.5containing 1% BSA (w/v at room temperature with gentle agitation.

Biotinylation of proteins: Biotin-NHS was dissolved in DD water at 50mg/l. 10 ul of this solution was added into maternal serum proteinsolution (5 mg/ml in 10 mM PB, pH8.5) and incubated for 3 hours on ashaker. 5 ul of ethanolamine was added to stop the reaction.Biotinylated proteins were diluted in 200 ul of TNB buffer and added toantibody arrays and incubated overnight at 4 C. Following three washesin TNT buffer, streptavidin-HRP was added and incubated for 30 minutesat room temperature. Antigen-Antibody interaction was detected usingCy5-tyramide fluorescence. Slides were scanned on a PE fluorescentscanner for quantification. Images of control and infected slides wereoverlayed using a image analysis program to generate a pseudocolorrepresentation for relative abundance. The results are shown in FIG. 9,which is a pseudocolor image of the protein array showing the binding ofcorresponding proteins with their antibodies. Green color representsinfected sample, red color represents control sample. Part II is anenlarged area of the array showing that calgranulin expression (green)is higher in the infected serum sample. Part III is a western blot ofcalgranulin IP showing similar increased expression in the infectedamniotic fluid sample.

Example 12 Further Analysis of Proteins Represented in the UniqueDiagnostic Signature of Infected Amniotic Fluid

It has been demonstrated that the SELDI-TOF profiles of control andinfected amniotic fluid show a unique signature in the mass range of10-12 KDa (FIGS. 1, 2 and 3), representative of positively infectedsample. The control and infected amniotic fluid resolved on a 1-D gel(FIG. 5) also shows bands in the mass range of 10-12 KDa that are moreabundant in the pooled or independent infected amniotic fluid samples.Isolation of these 1-D gel bands and further analysis using LCQ-MS asshown in FIG. 13, identified peptides representative of IGF-BR-1 andS-100 calcium binding proteins.

Western blot analysis of control and infected amniotic fluid using ananti-IGF-BP1 antibody as shown in FIG. 8, also demonstrates thedifferential expression of a proteolytic fragment (˜11 KDa) ininfection.

Sequencing of the amniotic fluid polypeptides also identified thepresence of IGF-BP1 and calgraulins in the infected amniotic fluid(Table 3).

The sequence of the identified novel proteolytic fragment of IGFBP-1 isshown in FIG. 12 (SEQ ID No: 1). In the Figure, the peptide sequencesfound in samples “0426seq_HI_(—)12” and “0425seq_HI-113” following 1-Dgel electrophoresis, trypsin digestion and MS/MS analysis of infectedamniotic fluid are shown in lower case (SEQ ID Nos: 2 and 3). Theproteolytic fragment of IGF-BP-1 detected in1-D gels (low molecularweight range, FIG. 5), Western blots (FIG. 6), and MS/MS analysis (FIG.13) of trypsin digested ˜10.5-12 KDa band from infected amniotic fluid,is represented in the region of the underlined sequence.

Indeed, MS/MS analysis and sequence search results demonstrated that theparent ion 434.89 in the mass spectrum shown in FIG. 13 represents anIGF-BP-1 sequence (RSPGSPEIR), which is also shown in the FIG. 12sequence map of the IGF-BP-1 proteolytic fragment. The parent ion1082.97 represents S-100 calcium binding proteins (i.e., Calgranulins Aand B), also independently identified by de novo sequencing of AF(Tables 2 and 3).

FIG. 14 shows mass spectrum for the 17.55-18.21 minute retention timepeak shown in FIG. 13. It is apparent that the dominate peak appears atmass 434.9.

FIG. 15 shows the MS/MS spectrum for the parent ion of the 434.9 peakshown in FIG. 14. Based on database search, the parent ion correspondsto a partial sequence of IGFBP-1.

Example 13 Diagnostic Profiles Characteristic of ChromosomalAneuploidies

The utility of proteomic profiling was examined to identify trisomy-21more accurately using maternal serum screening. This study was performedwith a panel of (control (n=6), trisomy-21 (n=6) and trisomy-18 (n=4),well-characterized maternal serum samples (matching amniotic fluidsamples for the same cases were tested by standard chromosomal mappingmethod and positively confirmed the presence of trisomies) and analyzedusing SELDI-TOF methodology as described above for the intrauterineinfection model.

FIG. 10 shows differential protein expression patterns in the maternalserum with unique profiles to distinguish trisomies. One microgram ofmaternal serum (after removal of albumin and immunoglobulins usingprotein separation columns, BioRad technologies) was used to performSELDI-TOF analysis of maternal serum extracts bound to chemicallydefined Normal Phase chip arrays as described in the methods. Wholespectrum collected at 235-laser intensity showing the differences in thepeak intensities. A) Control serum; B) trisomy-21 (Down's) serum; C)trisomy-18 serum. Detailed spectrum showing the differences in the 4-15KDa region unique for each case. Arrows indicate diagnostic peaks thatcan be used in a combination to formulate an algorithm to developdiagnostic screening tests.

This further illustrates detection of protein expression patterns invarious biological fluids (such as maternal serum) could identifyfetal-maternal conditions more accurately and in a non-invasiveapproach.

In conclusion, the data presented herein demonstrate that differentialexpression of proteins in the amniotic fluid as well as other biologicalfluids, such as serum, represents a valid approach for a rapid,non-invasive and accurate diagnosis, prognosis, and monitoring ofvarious maternal/fetal conditions and chromosomal aneuploidies.

Throughout the foregoing description the invention has been discussedwith reference to certain embodiments, but it is not so limited. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

All references cited throughout the description, and the referencescited therein, are hereby expressly incorporated by reference in theirentirety.

1. A method for predicting pre-term birth in a mammalian subject,comprising: (1) comparing the proteomic profile of a test sample of abiological fluid obtained from said mammalian subject with a proteomicprofile of a reference sample wherein the test sample proteomic profileand the reference sample proteomic profile comprise information of theexpression of at least one of the following: IGFBP-1, Gelsolinprecursor, Calgranulin A, Alpha-1-acid glycoprotein 1 precursor, andAlpha-1-antitrypsin precursor; or fragments or naturally occurringvariants, and; (2) predicting pre-term birth in said mammalian subjectif: (a) at least one unique expression signature of said proteomicprofiles differs in said test sample relative to said reference samplewherein said unique expression signature of said reference sampleproteomic profile comprises information of the expression of at leastone of the following: IGFBP-1, Gelsolin precursor, Calgranulin A,Alpha-1-acid glycoprotein 1 precursor, and Alpha-1-antitrypsinprecursor; or fragments or naturally occurring variants, and wherein thereference sample proteomic profile is a normal proteomic profile that isindicative of normal birth; or (b) at least one unique expressionsignature of said proteomic profiles is essentially the same in saidtest sample relative to said reference sample wherein said uniqueexpression signature of said reference sample proteomic profilecomprises information of the expression of at least one of thefollowing: IGFBP-1, Gelsolin precursor, Calgranulin A, Alpha-1-acidglycoprotein 1 precursor, and Alpha-1-antitrypsin precursor; orfragments or naturally occurring variants thereof, and wherein thereference sample proteomic profile is a proteomic profile that has beenpredetermined to be indicative of pre-term birth.
 2. The method of claim1 wherein said IGFBP-1 fragment comprises SEQ ID NO
 4. 3. The method ofclaim 1 wherein said test sample and said reference sample proteomicprofiles further comprise information of the expression of one or moreof the following polypeptides: Calgranulin B, Neutrophilgelatinase-associated lipocalin, Annexin II (Lipocortin II), andComplement C3 precursor; or fragments or naturally occurring variantsthereof.
 4. The method of claim 3 wherein said test sample and saidreference sample proteomic profiles further comprise information of theexpression of one or more of the following polypeptides: L-plastin(Lymphocyte cytosolic protein 1), Profilin I, Alpha-1-acid glycoprotein2 precursor, Alpha-1-antichymotrypsin precursor, Ceruloplasminprecursor, Apolipoprotein A-I precursor (Apo-AI), 14-3-3 proteinzeta/delta plasma, Vitamin D-binding protein precursor, Serotransferrin,Squamous cell carcinoma antigen 1, 14-3-3 protein sigma (Stratifin),Cystatin A (Stefin A), Fibronectin precursor, Squamous cell carcinomaantigen 2, and Azurocidin; or fragments or naturally occurring variants.5. The method of claim 4 wherein the proteomic profiles are representedin the form of mass spectra.
 6. The method of claim 5 wherein saidmammalian subject is a primate.
 7. The method of claim 6 wherein saidprimate is human.
 8. The method of claim 1 wherein the biological fluidis amniotic fluid or maternal serum.
 9. The method of claim 1 whereinsaid proteomic profiles comprise information of the expression of atleast 10 proteins.
 10. The method of claim 1 wherein said proteomicprofiles comprise information of the expression of at least 20 proteins.11. The method of claim 1 wherein said proteomic profiles compriseinformation of the expression of at least 50 proteins.
 12. A method forpredicting pre-term birth in a mammalian subject, comprising: (1)comparing the proteomic profile of a test sample of a biological fluidwith a proteomic profile of a reference sample wherein said test sampleproteomic profile and said reference sample proteomic profile compriseinformation of the expression of IGFBP-1 or fragments or naturallyoccurring variants thereof, and; (2) predicting pre-term birth in saidmammalian subject if: (A) at least one unique expression signature ofsaid proteomic profiles differs in said test sample relative to saidreference sample, wherein said unique expression signature of saidreference sample proteomic profile comprises information of theexpression of IGFBP-1 or fragments or naturally occurring variantsthereof; and wherein the reference sample proteomic profile is a normalproteomic profile that is indicative of normal birth; or (B) at leastone unique expression signature of said proteomic profiles isessentially the same in said test sample relative to said referencesample wherein said unique expression signature of said reference sampleproteomic profile comprises information of the expression of IGFBP-1, orfragments or naturally occurring variants thereof and wherein thereference sample proteomic profile is a proteomic profile that has beenpredetermined to beindicative of pre-term birth.
 13. The method of claim1 wherein said IGFBP-1 fragment comprises SEQ ID NO
 4. 14. The method ofclaim 13 wherein the proteomic profiles are represented in the form ofmass spectra.
 15. The method of claim 12 wherein said mammalian subjectis a primate.
 16. The method of claim 15 wherein said primate is human.17. The method of claim 12 wherein the biological fluid is amnioticfluid or maternal serum.
 18. The method of claim 12 wherein saidproteomic profiles comprise information of the expression of at least 10proteins.
 19. The method of claim 12 wherein said proteomic profilescomprise information of the expression of at least 20 proteins.
 20. Themethod of claim 12 wherein said proteomic profiles comprise informationof the expression of at least 50 proteins.